Clostridium difficile is a spore-forming anaerobic bacillus that causes severe colitis with high mortality. The incidence and severity of C. difficile infections (CDIs) are both increasing at an alarming rate. Treatment is associated with unacceptable failure and relapse rates and no vaccine has been licensed. While disease is strongly dependent on the elaboration of cytotoxins, the mechanisms required for colonization are essentially unknown. This knowledge gap inhibits the development of novel strategies to prevent acquisition of infection. Type IV pili (T4Ps) represent the most widespread class of pili, broadly distributed among diverse phylogenetic divisions including the Clostridiales. C. difficile genomes have the complete set of T4P biogenesis genes including several pilin or pilin-like proteins. As T4Ps play essential roles in the colonization and adherence to tissues of numerous animals during the pathogenesis of multiple infectious diseases, we aim to test the hypothesis that T4Ps play a role in colonization and disease in CDI. To do so, we will generate a prepilin peptidase mutant of an epidemic C. difficile strain, complement the mutant, confirm its phenotype and compare the ability of the wild type, mutant and complemented strain in a murine model of CDI. The structures of several T4P pilin proteins have been solved and revealed significant insights into the biogenesis and function of these important proteins. These insights have been enhanced by solving the structures of distantly related pilin and pilin-like proteins. We have purified to homogeneity two C. difficile pilin proteins. We have obtained high-quality 2D NMR data from one of these proteins and have crystallized the other. We aim to determine the structures of both proteins providing critical information about the most divergent members of this class thus far investigated. The proposed studies will establish whether T4Ps are important C. difficile colonization and virulence factors and deepen our appreciation of the structure and biogenesis of these ubiquitous pili.